Work described herein was supported by the National Institutes of Health (RO1-EY29410 to J.M.G, and NIH CORE Grant P30-EY08098 to the Department of Ophthalmology); the UPMC Immune Transplant &#38; Therapy Center (to L.L.L. and J.M.G.); and the E. Ronald Salvitti Chair in Ophthalmology Research (to J.M.G.). Additional support was received from the Wiegand Fellowship in Ophthalmology (to L.L.L), the Eye &#38; Ear Foundation of Pittsburgh, and an unrestricted grant from Research to Prevent Blindness, New York, NY. Authors also wish to thank Amanda Platt for technical assistance and Dr. Hugh Hammer and the aquatics staff for excellent animal care support.

L.L.L. is the co-inventor on US Patent #9,458,428, which describes an expedited method to derive retinal pigment epithelium from human pluripotent stem cells; this is unrelated to the content herein. J.M.G. and G.B.F. have nothing to disclose.

This protocol describes methodology to genetically ablate the RPE and study mechanisms of degeneration and regeneration in larval-aged zebrafish. This protocol has also been successfully performed in adult zebrafish3 but with less extensive characterization, which is why larvae are the focus here. Critical aspects of this part of the protocol (steps 1-4) include: 1) adding 1.5x PTU to embryos prior to the onset of melanogenesis, 2) dechorionating PTU-treated embryos on 2-3 dpf, 3) careful screenin...

The methodology described herein details a protocol to genetically ablate the retinal pigment epithelium (RPE) utilizing larval zebrafish. The RPE extends over the back of the eye and resides between the stratified layers of the neural retina and the layer of vasculature constituting the choroid. Trophic support, absorption of phototoxic light, and maintenance of visual cycle proteins are only some of the critical functions the RPE performs that are essential for sustaining the health and integrity of these adjacent tissues1. Damage to mammalian RPE is reparable when lesions are small2; however, damage suffered by larger injuries or progressive degenerative disease is irreversible. In humans, RPE degenerative diseases (e.g.,&#160;age-related macular degeneration (AMD) and Stargardt disease) lead to permanent vision loss and, with few treatment options available, decreased patient&#160;quality of life. The limited ability for mammalian RPE to self-repair has created a knowledge gap in the field of RPE regenerative processes. Given the robust regenerative capacity of the zebrafish across many different tissue types, this protocol was developed to establish an in vivo vertebrate system to facilitate studies on intrinsically regenerating RPE and uncover mechanisms that drive that response. Using the ablation paradigm outlined here, the canonical Wnt signaling pathway3, the mTOR pathway4, and immune-related responses5 have been identified as critical mediators of RPE regeneration, likely with overlapping functions.
In this genetic ablation paradigm, Tg(rpe65a:nfsB-eGFP)3 zebrafish express the bacterial-derived nitroreductase (NTR/nfsB) gene6 fused to eGFP under control of the RPE enhancer element, rpe65a7. Ablation is achieved by adding the prodrug, metronidazole (MTZ), to system water housing zebrafish. Intracellular activation of MTZ by nitroreductase results in DNA crosslinking and apoptosis in NTR/nfsB-expressing cells8,9. This technology has been widely used in zebrafish to ablate cells of the retina10,11,12,13 and other tissues8. Together, these elements enable targeted expression (rpe65a) of an inducible cell ablation methodology (NTR/MTZ)8,9 and a fluorescent marker (eGFP) for visualization.
Other interesting in vivo models also exist that can be used to study the regenerative potential of the RPE14. These are broad and include RPE-to-retina transdifferentiation post-retinectomy in amphibians, in which RPE cells lost to retinal regrowth are replaced15,16; RPE restoration post-injury in the &#34;super healing&#34; MRL/MpJ mouse17; and exogenous stimulation of RPE proliferation in a rat model of spontaneous RPE and retinal degeneration18, among others. In vitro models, such as adult human RPE stem cells (RPESCs)19 have also been developed. These models are all valuable tools working to uncover the cellular processes related to RPE regeneration (e.g.,&#160;proliferation, differentiation, etc.); however, the zebrafish is unique in its capacity for intrinsic RPE repair post-ablation.
While the methodology here is written to focus on understanding the mechanisms driving RPE regeneration, the Tg(rpe65a:nfsB-eGFP) line and this genetic ablation protocol could be utilized to study other cellular processes such as RPE apoptosis, RPE degeneration, and the effect of RPE injury on adjacent retinal and vascular tissues. The ablation protocol can also be modified to include pharmacological manipulation, which is a convenient preliminary strategy to screen signaling pathways of interest. For example, blocking the canonical Wnt pathway using Inhibitor of Wnt Response-1 (IWR-1)20, has been shown to impair RPE regeneration3. This was repeated here to guide users through a pharmacological manipulation experiment and serve as proof-of-concept to validate a MATLAB script (RpEGEN) created to quantify RPE regeneration based on recovery of pigmentation. Like the transgenic line and ablation protocol, the RpEGEN scripts are adaptable and could be used to quantify other markers/cellular processes within the RPE.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Lab Material/Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI)</td>
			<td>Millipore Sigma</td>
			<td>D9542</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plates</td>
			<td>Fisher Scientific</td>
			<td>07-200-83</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical Polypropylene Centrifuge Tubes</td>
			<td>Fisher Scientific</td>
			<td>05-539-13</td>
			<td>Catalog number is for 50 mL tubes</td>
		</tr>
		<tr>
			<td>Diamond tip scribing pen</td>
			<td>Fisher Scientific</td>
			<td>50-254-51</td>
			<td>Manufactured by Electron Microscopy Sciences, items similar to this part number are adequate</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO) &#8805;99.7 %</td>
			<td>Fisher Scientific</td>
			<td>BP231</td>
			<td>Check instiutional chemical waste disposal requirements</td>
		</tr>
		<tr>
			<td>Embryo incubator (large)</td>
			<td>Fisher Scientific</td>
			<td>3720A</td>
			<td></td>
		</tr>
		<tr>
			<td>Embryo incubator (mini/tabletop)</td>
			<td>Labnet</td>
			<td>I5110A</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence stereo microscope</td>
			<td>Zeiss</td>
			<td>Axio Zoom.V16</td>
			<td>Or similar, with 488 nm excitation laser/filter</td>
		</tr>
		<tr>
			<td>Glass Pasteur pipette</td>
			<td>Fisher Scientific</td>
			<td>13-678-4</td>
			<td>Manufactured by Corning, non-sterile</td>
		</tr>
		<tr>
			<td>InSolution Wnt Antagonist I, IWR-1-endo</td>
			<td>Millipore Sigma</td>
			<td>5.04462</td>
			<td>Manufactured by Calbiochem; 25 mM in DMSO; check instiutional chemical waste disposal requirements</td>
		</tr>
		<tr>
			<td>Methylene blue (powder)</td>
			<td>Fisher Scientific</td>
			<td>BP117-100</td>
			<td>Also available as a premade aqeuous solution</td>
		</tr>
		<tr>
			<td>Metronidazole (MTZ)</td>
			<td>Millipore Sigma</td>
			<td>M3761</td>
			<td>Check instiutional chemical waste disposal requirements</td>
		</tr>
		<tr>
			<td>N-phenylthiourea (PTU)</td>
			<td>Millipore Sigma</td>
			<td>P7629</td>
			<td>Check instiutional chemical waste disposal requirements</td>
		</tr>
		<tr>
			<td>Paraformaldehyde (16 % w/v) methanol free</td>
			<td>Fisher Scientific</td>
			<td>AA433689M</td>
			<td>Chemical waste, proper disposal required</td>
		</tr>
		<tr>
			<td>Petri dishes</td>
			<td>Fisher Scientific</td>
			<td>FB0875712</td>
			<td>10 cm diameter</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (powder packets)</td>
			<td>Millipore Sigma</td>
			<td>P3813</td>
			<td>Used to make 10 X PBS stock</td>
		</tr>
		<tr>
			<td>Pronase</td>
			<td>Millipore Sigma</td>
			<td>PRON-RO</td>
			<td></td>
		</tr>
		<tr>
			<td>Shaking incubator</td>
			<td>Benchmark</td>
			<td>H2010</td>
			<td>Used for incubating MTZ for 1 hour at 37 degrees Celcius</td>
		</tr>
		<tr>
			<td>Stereo microscope</td>
			<td>Leica</td>
			<td>S9i</td>
			<td>Or similar, with transmitted light illumination</td>
		</tr>
		<tr>
			<td>Student Dumont #5 forceps</td>
			<td>Fine Science Tools</td>
			<td>91150-20</td>
			<td>Fine-tipped forceps for manual dechorionation</td>
		</tr>
		<tr>
			<td>Tabletop rotator/shaker</td>
			<td>Scilogex</td>
			<td>SK-D1807-E</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfer pipette</td>
			<td>Millipore Sigma</td>
			<td>Z135003</td>
			<td>3.2 mL bulb draw, non-sterile</td>
		</tr>
		<tr>
			<td>Tricaine methanesulfonate (MS-222)</td>
			<td>Pentair</td>
			<td>TRS1, TRS2, TRS5</td>
			<td>Also available from Fisher Scientific (NC0342409)</td>
		</tr>
		<tr>
			<td>VECTASHIELD Antifade Mounting Medium with DAPI</td>
			<td>Vector Laboratories</td>
			<td>H-1200</td>
			<td></td>
		</tr>
		<tr>
			<td>Software Material</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FIJI (Fiji is Just ImageJ)</td>
			<td>FIJI (Fiji is Just ImageJ)</td>
			<td>https://imagej.net/software/fiji/</td>
			<td>Version: 2.0.0-rc-69/1.52p; Build: 269a0ad53f; Plugin needed: Bio-Formats</td>
		</tr>
		<tr>
			<td>GRAMM examples and how-tos</td>
			<td>MathWorks</td>
			<td>https://www.mathworks.com/matlabcentral/fileexchange/54465-gramm-complete-data-visualization-toolbox-ggplot2-r-like.</td>
			<td></td>
		</tr>
		<tr>
			<td>MATLAB</td>
			<td>MathWorks</td>
			<td>https://www.mathworks.com/products/get-matlab.html</td>
			<td>Toolboxes needed to run RpEGEN: Image Processing Toolbox, Curve Fitting Toolbox, Statistics and Machine Learning Toolbox</td>
		</tr>
		<tr>
			<td>MATLAB support</td>
			<td>MathWorks</td>
			<td>https://www.mathworks.com/support.html</td>
			<td></td>
		</tr>
	</tbody>
</table>

nitroreductase/metronidazole-mediated ablation and a matlab platform (rpegen) for studying regeneration of the zebrafish retinal pigment epithelium

The retinal pigment epithelium (RPE) resides at the back of the eye and performs functions essential for maintaining the health and integrity of adjacent retinal and vascular tissues. At present, the limited reparative capacity of mammalian RPE, which is restricted to small injuries, has hindered progress to understanding in vivo RPE regenerative processes. Here, a detailed methodology is provided to facilitate the study of in vivo RPE repair utilizing the zebrafish, a&#160;vertebrate model capable of robust tissue regeneration. This protocol describes a transgenic nitroreductase/metronidazole (NTR/MTZ)-mediated injury paradigm (rpe65a:nfsB-eGFP), which results in ablation of the central two-thirds of the RPE after 24 h treatment with MTZ, with subsequent tissue recovery. Focus is placed on RPE ablations in larval zebrafish and methods for testing the effects of pharmacological compounds on RPE regeneration are also outlined. Generation and validation of RpEGEN, a MATLAB script created to automate quantification of RPE regeneration based on pigmentation, is also discussed. Beyond active RPE repair mechanisms, this protocol can be expanded to studies of RPE degeneration and injury responses as well as the effects of RPE damage on adjacent retinal and vascular tissues, among other cellular and molecular processes. This zebrafish system holds significant promise in identifying genes, networks, and processes that drive RPE regeneration and RPE disease-related mechanisms, with the long-term goal of applying this knowledge to mammalian systems and, ultimately, toward therapeutic development.

Inhibiting the canonical Wnt signaling pathway is known to significantly impair zebrafish RPE regeneration using the genetic ablation paradigm (rpe65a:nfsB-eGFP) and pharmacological manipulation methodology (IWR-1) described in the protocol3. This experiment was repeated here to validate an automated method for quantifying zebrafish RPE regeneration based on pigmentation. The results summarized below encompassed all steps of the protocol, from the day of fertilization (0 dpf) to quantific...

Watch this Scientific Journal Video about Nitroreductase/Metronidazole-Mediated Ablation and a MATLAB Platform RpEGEN for Studying Regeneration of the Zebrafish Retinal Pigment Epithelium at JoVE.com

Nitroreductase/Metronidazole-Mediated Ablation and a MATLAB Platform RpEGEN for Studying Regeneration of the Zebrafish Retinal Pigment Epithelium

This protocol describes the methodology to genetically ablate the retinal pigment epithelium (RPE) using a transgenic zebrafish model. Adapting the protocol to incorporate signaling pathway modulation using pharmacological compounds is extensively detailed. A MATLAB platform for quantifying RPE regeneration based on pigmentation was developed and is presented and discussed.

Nitroreductase/Metronidazole-Mediated Ablation and a MATLAB Platform (RpEGEN) for Studying Regeneration of the Zebrafish Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) resides at the back of the eye and performs functions essential for maintaining the health and integrity of adjacent ...

nitroreductasemetronidazole-mediated-ablation-matlab-platform-rpegen

Research

JoVE Journal

Biology

2.4K Views.  University of Pittsburgh School of Medicine. This protocol describes the methodology to genetically ablate the retinal pigment epithelium (RPE) using a transgenic zebrafish model. Adapting the protocol to incorporate signaling pathway modulation using pharmacological compounds is extensively detailed. A MATLAB platform for quantifying RPE regeneration based on pigmentation was developed and is presented and discussed.

Video: Nitroreductase/Metronidazole-Mediated Ablation and a MATLAB Platform RpEGEN for Studying Regeneration of the Zebrafish Retinal Pigment Epithelium

A System for Culturing Iris Pigment Epithelial Cells to Study Lens Regeneration in Newt

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, heart, the jaw 1. Specifically, newts are unique for its lens regeneration capability. Upon lens removal, IPE cells of the dorsal iris transdifferentiate to lens cells and eventually form a new lens in about a month 2,3. This property of regeneration is never exhibited by the ventral iris cells. The regeneration potential of the iris cells can be studied by making transplants of the in vitro cultured IPE cells. For the culture, the dorsal and ventral iris cells are first isolated from the eye and cultured separately for a time period of 2 weeks (Figure 1). These cultured cells are reaggregated and implanted back to the newt eye. Past studies have shown that the dorsal reaggregate maintains its lens forming capacity whereas the ventral aggregate does not form a lens, recapitulating, thus the in vivo process (Figure 2) 4,5. This system of determining regeneration potential of dorsal and ventral iris cells is very useful in studying the role of genes and proteins involved in lens regeneration.

In newt, the lens regenerates always from the dorsal iris by transdifferentiation of the iris pigmented epithelial cells (IPEs). Here we describe a procedure to culture dorsal and ventral newt IPE cells and their implantation to the newt eye. The implanted cells are then studied by tissue sectioning and immunohistochemistry.

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, ...

Laser Ablation of the Zebrafish Pronephros to Study Renal Epithelial Regeneration

Acute kidney injury (AKI) is characterized by high mortality rates from deterioration of renal function over a period of hours or days that culminates in renal failure1. AKI can be caused by a number of factors including ischemia, drug-based toxicity, or obstructive injury1. This results in an inability to maintain fluid and electrolyte homeostasis. While AKI has been observed for decades, effective clinical therapies have yet to be developed. Intriguingly, some patients with AKI recover renal functions over time, a mysterious phenomenon that has been only rudimentally characterized1,2. Research using mammalian models of AKI has shown that ischemic or nephrotoxin-injured kidneys experience epithelial cell death in nephron tubules1,2, the functional units of the kidney that are made up of a series of specialized regions (segments) of epithelial cell types3. Within nephrons, epithelial cell death is highest in proximal tubule cells. There is evidence that suggests cell destruction is followed by dedifferentiation, proliferation, and migration of surrounding epithelial cells, which can regenerate the nephron entirely1,2. However, there are many unanswered questions about the mechanisms of renal epithelial regeneration, ranging from the signals that modulate these events to reasons for the wide variation of abilities among humans to regenerate injured kidneys.
The larval zebrafish provides an excellent model to study kidney epithelial regeneration as its pronephric kidney is comprised of nephrons that are conserved with higher vertebrates including mammals4,5. The nephrons of zebrafish larvae can be visualized with fluorescence techniques because of the relative transparency of the young zebrafish6. This provides a unique opportunity to image cell and molecular changes in real-time, in contrast to mammalian models where nephrons are inaccessible because the kidneys are structurally complex systems internalized within the animal. Recent studies have employed the aminoglycoside gentamicin as a toxic causative agent for study of AKI and subsequent renal failure: gentamicin and other antibiotics have been shown to cause AKI in humans, and researchers have formulated methods to use this agent to trigger kidney damage in zebrafish7,8. However, the effects of aminoglycoside toxicity in zebrafish larvae are catastrophic and lethal, which presents a difficulty when studying epithelial regeneration and function over time. Our method presents the use of targeted cell ablation as a novel tool for the study of epithelial injury in zebrafish. Laser ablation gives researchers the ability to induce cell death in a limited population of cells. Varying areas of cells can be targeted based on morphological location, function, or even expression of a particular cellular phenotype. Thus, laser ablation will increase the specificity of what researchers can study, and can be a powerful new approach to shed light on the mechanisms of renal epithelial regeneration. This protocol can be broadly applied to target cell populations in other organs in the zebrafish embryo to study injury and regeneration in any number of contexts of interest.

Acute kidney injury (AKI) in humans is a common clinical problem caused by damage to the epithelial cells that comprise kidney nephrons, and AKI is associated with high mortality rates of 50-70%1. Following epithelial cell destruction, nephrons have a limited ability to regenerate, though the mechanisms and limitations that guide this phenomenon remain poorly understood. In this video article, we describe our technique for targeted laser ablation of kidney nephron cells in the zebrafish embryo kidney, or pronephros. Our new method can be used to complement nephrotoxicity-induced models of AKI and gain a high-resolution understanding of the cell and molecular alterations that are associated with epithelial regeneration in the kidney nephron.

Acute kidney injury (AKI) is characterized by high mortality rates from deterioration of renal function over a period of hours or days that culminates in ...

MicroRNA Expression Profiles of Human iPS Cells, Retinal Pigment Epithelium Derived From iPS, and Fetal Retinal Pigment Epithelium

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human iPS cells (iPS-RPE), and fetal RPE. The protocols include collection of RNA for analysis by microarray, and the analysis of microarray data to identify miRNAs that are differentially expressed among three cell types. The methods for culture of iPS cells and fetal RPE are explained. The protocol used for differentiation of RPE from human iPS is also described. The RNA extraction technique we describe was selected to allow maximal recovery of very small RNA for use in a miRNA microarray. Finally, cellular pathway and network analysis of microarray data is explained. These techniques will facilitate the comparison of the miRNA profiles of three different cell types.

The microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human induced-pluripotent stem (iPS) cells (iPS-RPE), and fetal RPE, were compared.

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, ...

Methylnitrosourea (MNU)-induced Retinal Degeneration and Regeneration in the Zebrafish: Histological and Functional Characteristics

Retinal degenerative diseases, e.g. retinitis pigmentosa, with resulting photoreceptor damage account for the majority of vision loss in the industrial world. Animal models are of pivotal importance to study such diseases. In this regard the photoreceptor-specific toxin N-methyl-N-nitrosourea (MNU) has been widely used in rodents to pharmacologically induce retinal degeneration. Previously, we have established a MNU-induced retinal degeneration model in the zebrafish, another popular model system in visual research.
A fascinating difference to mammals is the persistent neurogenesis in the adult zebrafish retina and its regeneration after damage. To quantify this observation we have employed visual acuity measurements in the adult zebrafish. Thereby, the optokinetic reflex was used to follow functional changes in non-anesthetized fish. This was supplemented with histology as well as immunohistochemical staining for apoptosis (TUNEL) and proliferation (PCNA) to correlate the developing morphological changes.
In summary, apoptosis of photoreceptors occurs three days after MNU treatment, which is followed by a marked reduction of cells in the outer nuclear layer (ONL). Thereafter, proliferation of cells in the inner nuclear layer (INL) and ONL is observed. Herein, we reveal that not only a complete histological but also a functional regeneration occurs over a time course of 30 days. Now we illustrate the methods to quantify and follow up zebrafish retinal de- and regeneration using MNU in a video-format.

Methylnitrosourea MNU-induced Retinal Degeneration and Regeneration in the Zebrafish: Histological and Functional Characteristics

Herein we demonstrate quantification of retinal de- and regeneration and its impact on visual function using N-methyl-N-nitrosourea in the adult zebrafish. Loss of visual acuity and decreased photoreceptor numbers were followed by proliferation in the inner nuclear layer. Complete morphological and functional regeneration occurred 30 days after the initial treatment.

Retinal degenerative diseases, e.g. retinitis pigmentosa, with resulting photoreceptor damage account for the majority of vision loss in the industrial ...

Primary Cell Cultures from the Mouse Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) is a highly polarized multi-functional epithelium that is located between the neural retina and the choroid of the eye. It is a single sheet of pigmented cells that are hexagonally packed and connected by tight junctions. The main functions of the RPE include absorption of light, phagocytosis of the shed photoreceptor outer segments, spatial buffering of ions, transport of nutrients, ions and water as well as active involvement in the visual cycle. With such important and diverse functions, it is critically important to study the biology of RPE cells. A number of RPE cell lines have been established; however, passaged and immortalized cells are known to quickly lose some of the morphological and physiological characteristics of natural RPE cells. Thus, primary cells are more suitable for studying different aspects of RPE cell biology and function. Mouse primary RPE cell culture is very useful to researchers since mouse models are widely used in biological studies, however collecting RPE cells from mouse is also very challenging due to their small size. Here, we present a protocol for establishing primary mouse RPE cell cultures which includes enucleation and dissection of the eyes and isolation of the RPE sheets to yield the cells for culturing. This method enables efficient cell recovery. The RPE cells obtained from two mice&#160;can reach confluency on one 12 mm polyester membrane insert pre-loaded in culture plate after one week of culture and display some of the original properties of bona fide RPE cells such as hexagonal shape and pigmentation after two weeks of culture.

The retinal pigment epithelium (RPE) is a multi-functional epithelium of the eye. Here we present a protocol to establish primary cell cultures derived from the murine RPE.

The retinal pigment epithelium (RPE) is a highly polarized multi-functional epithelium that is located between the neural retina and the choroid of the ...

Efficient and Consistent Generation of Retinal Pigment Epithelium/Choroid Flatmounts from Human Eyes for Histological Analysis

The retinal pigment epithelium (RPE) and retina are functionally and structurally connected tissues that work together to regulate light perception and vision. Proteins on the RPE apical surface are tightly associated with proteins on the photoreceptor outer segment surface, making it difficult to consistently separate the RPE from the photoreceptors/retina. We developed a method to efficiently separate the retina from the RPE of human eyes to generate complete RPE/choroid and retina flatmounts for separate cellular analysis of the photoreceptors and RPE cells. An intravitreal injection of a high-osmolarity solution of D-mannitol, a sugar not transported by the RPE, induced the separation of the RPE and retina across the entire posterior chamber without causing damage to the RPE cell junctions. No RPE patches were observed attached to the retina. Phalloidin labeling of actin showed RPE shape preservation and allowed morphometric analysis of the entire epithelium. An artificial intelligence (AI)-based software was developed to accurately recognize and segment the RPE cell borders and quantify 30 different shape metrics. This dissection method is highly reproducible and can be easily extended to other animal models.

We describe a method to efficiently separate retinal pigment epithelium (RPE) from the retina in human eyes and generate whole RPE/choroid flatmounts for histological and morphometric analyses of the RPE.

The retinal pigment epithelium (RPE) and retina are functionally and structurally connected tissues that work together to regulate light perception and ...

Caudal Peduncle Cryoinjury in Adult Zebrafish

A Cryoinjury Model for Studying Skeletal Muscle Regeneration of the Caudal Peduncle in Adult Zebrafish

Skeletal muscle undergoes renewal and restoration after minor injury through the activation of satellite-like stem cells. Severe injuries of the musculature often lead to fibrosis in humans. In comparison to mammals, zebrafish possess a higher innate capacity for organ regeneration, providing a powerful model for studying tissue restoration after extensive damage to the organ. Here, a cryoinjury model is described to induce profound damage to four myomeres of the caudal peduncle in adult zebrafish. A custom-made cryoprobe was designed to fit the body shape and reproducibly injure the lateral musculature from the skin to the midline. Importantly, the body integrity remained intact, and the fish continued their swimming activity. Changes to the skeletal muscle were assessed by histological staining and fluorescence staining of sarcomeric proteins on tissue sections. This method will open up new avenues of research aiming to understand how the degeneration of the skeletal muscle induces reparative responses and, thus, the reactivation of the myogenic program in adult zebrafish.

Author Spotlight: A Cryoinjury Model for Studying Skeletal Muscle Regeneration of the Caudal Peduncle in Adult Zebrafish  

This protocol describes a cryoinjury model to induce profound damage of several caudal myomeres in adult zebrafish. This method provides a new approach for studying skeletal muscle regeneration after severe loss of tissue in non-mammalian vertebrates.

Skeletal muscle undergoes renewal and restoration after minor injury through the activation of satellite-like stem cells. Severe injuries of the ...

Modeling and Analyzing Retinal Degeneration

LipidUNet-Machine Learning-Based Method of Characterization and Quantification of Lipid Deposits Using iPSC-Derived Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) is a monolayer of hexagonal cells located at the back of the eye. It provides nourishment and support to photoreceptors and choroidal capillaries, performs phagocytosis of photoreceptor outer segments (POS), and secretes cytokines in a polarized manner for maintaining the homeostasis of the outer retina. Dysfunctional RPE, caused by mutations, aging, and environmental factors, results in the degeneration of other retinal layers and causes vision loss. A hallmark phenotypic feature of degenerating RPE is intra and sub-cellular lipid-rich deposits. These deposits are a common phenotype across different retinal degenerative diseases. To reproduce the lipid deposit phenotype of monogenic retinal degenerations in vitro, induced pluripotent stem cell-derived RPE (iRPE) was generated from patients&#39; fibroblasts. Cell lines generated from patients with Stargardt and Late-onset retinal degeneration (L-ORD) disease were fed with POS for 7 days to replicate RPE physiological function, which caused POS phagocytosis-induced pathology in these diseases. To generate a model for age-related macular degeneration (AMD), a polygenic disease associated with alternate complement activation, iRPE was challenged with alternate complement anaphylatoxins. The intra and sub-cellular lipid deposits were characterized using Nile Red, boron-dipyrromethene (BODIPY), and apolipoprotein E (APOE). To quantify the density of lipid deposits, a machine learning-based software, LipidUNet, was developed. The software was trained on maximum-intensity projection images of iRPE on culture surfaces. In the future, it will be trained to analyze three-dimensional (3D) images and quantify the volume of lipid droplets. The LipidUNet software will be a valuable resource for discovering drugs that decrease lipid accumulation in disease models.

Author Spotlight: Unraveling the Pathogenesis of Age-Related Macular Degeneration and Discovering Potential Therapies 

Degenerative eye diseases that affect the retinal pigment epithelium layer of the eye have monogenic and polygenic origins. Several disease models and a software application, LipidUNet, have been developed to study mechanisms of disease, as well as potential therapeutic interventions.

The retinal pigment epithelium (RPE) is a monolayer of hexagonal cells located at the back of the eye. It provides nourishment and support to ...

Staining of Sub-Retinal Pigment Epithelium (RPE) Deposits

A Cost-Effective Method for Isolating Primary RPE Cells from Porcine Eyes

Isolation of Primary Porcine Retinal Pigment Epithelial Cells for In Vitro Modeling

The retinal pigment epithelium (RPE) is a crucial monolayer in the outer retina responsible for supporting photoreceptors. RPE degeneration commonly occurs in diseases marked by progressive vision loss, such as age-related macular degeneration (AMD). Research on AMD often relies on human donor eyes or induced pluripotent stem cells (iPSCs) to represent the RPE. However, these RPE sources require extended differentiation periods and substantial expertise for culturing. Additionally, some research institutions, particularly those in rural areas, lack easy access to donor eyes. While a commercially available immortalized RPE cell line (ARPE-19) exists, it lacks essential in vivo RPE features and is not widely accepted in many ophthalmology research publications. There is a pressing need to obtain representative primary RPE cells from a more readily available and cost-effective source. This protocol elucidates the isolation and subculture of primary RPE cells obtained post-mortem from porcine eyes, which can be sourced locally from commercial or academic suppliers. This protocol necessitates common materials typically found in tissue culture labs. The result is a primary, differentiated, and cost-effective alternative to iPSCs, human donor eyes, and ARPE-19 cells.

Author Spotlight: Modeling Retinal Pathologies with Enhanced RPE Cell-Based Disease Models

This protocol outlines the procedure for obtaining and culturing primary retinal pigment epithelial (RPE) cells from locally sourced porcine eyes. These cells serve as a high-quality alternative to stem cells and are suitable for in vitro retinal research.

The retinal pigment epithelium (RPE) is a crucial monolayer in the outer retina responsible for supporting photoreceptors. RPE degeneration commonly ...